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NO39 - 2

Effect of Tobramycin Supplementation to Medium on Isolation of Flavobacterium psychrophilum from Ayu Plecoglossus altivelis

<>Akira Kumagai1, 3*, Chihaya Nakayasu1 and Norihisa Oseko2<>1Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency,
Tamaki, Mie 519-0423, Japan
2National Research Institute of Aquaculture, Fisheries Research Agency, Nansei,
Mie 516-0193, Japan
3Present address:Miyagi Prefectural Freshwater Fisheries Experimental Station,
Taiwa, Miyagi 981-3625, Japan
(Received July 24, 2003)
ABSTRACT - The effect of the addition of the aminoglycoside antibiotic tobramycin to the culture medium on selective isolation of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, from ayu Plecoglossus altivelis was examined. The minimum inhibitory concentrations of tobramycin against all 37 strains of F. psychrophilum tested were more than 6.3 μg/mL. The addition of tobramycin to medium at a concentration of 5 μg/mL was a better method for the isolation of F. psychrophilum, because the overgrowth of other bacteria was inhibited and the isolation rates of F. psychrophilum were improved. Especially, the use of tobramycin-supplemented medium could be effective in the examination of the external organs of carrier fish, in which viable bacterial counts of F. psychrophilum are lower than those of other bacteria.

Key words: tobramycin, Flavobacterium psychrophilum, selective isolation, bacterial coldwater disease, Plecoglossus altivelis

Kudoa thyrsites from Japanese Flounder and Kudoa lateolabracis n. sp. from Chinese Sea Bass:Causative Myxozoans of Post-Mortem Myoliquefaction

<>Hiroshi Yokoyama1*, Christopher M. Whipps2, Michael L. Kent2,Kaori Mizuno3 and Hidemasa Kawakami3<>1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Tokyo 113-8657, Japan
2Center for Fish Disease Research, Department of Microbiology, Oregon State
University, Corvallis, Oregon 97331-3404, USA
3Ehime Prefectural Fish Disease Control Center, Uwajima,
Ehime 798-0087, Japan
(Received November 17, 2003)
ABSTRACT - Post-mortem myoliquefaction caused by myxozoans of the genus Kudoa was observed in Japanese flounder Paralichthys olivaceus and Chinese sea bass Lateolabrax sp. cultured in Japan. Morphological and molecular analyses of the myxozoan from Japanese flounder identified it as Kudoa thyrsites, which has been described from various marine fishes in different oceans. The parasite from Chinese sea bass was similar to K. thyrsites, having stellate spores with one polar capsule larger than the other three. However, the spore size was smaller than that of K. thyrsites. The small subunit rDNA sequence from the Kudoa sp. of Chinese sea bass was distinct from that of K. thyrsites and phylogenetic analysis placed it as an outlier to K. thyrsites.
Therefore, we describe the myxozoan from Chinese sea bass as Kudoa lateolabracis n. sp.

Key words: myxozoan, Kudoa thyrsites, Kudoa lateolabracis, Paralichthys olivaceus, Lateolabrax sp., Japanese flounder, Chinese sea bass, myoliquefaction

Identification and Characterization of a Fimbrial Gene Cluster of Edwardsiella tarda Expressing Mannose-resistant Hemagglutination

<>Takamitsu Sakai1, Kinya Kanai2*, Kiyoshi Osatomi2 and Kazuma Yoshikoshi2<>1Graduate School of Science and Technology, Nagasaki University,
Nagasaki, 852-8521, Japan
2Faculty of Fisheries, Nagasaki University, Nagasaki,
852-8521, Japan
(Received December 5, 2003)
ABSTRACT - We determined the nucleotide sequence of a 8.6 kb DNA region containing etfA encoding a putative fimbrial major subunit from chromosomal DNA of Edwardsiella tarda KG8401 which expresses mannose-resistant hemagglutination (MRHA). This region contained three novel genes, etfBCD, at the downstream region of etfA. The deduced amino acid sequences of EtfABCD contained conserved fimbrial domains; fimbrial protein, fimbrial chaperone, fimbrial usher and fimbrial protein, respectively. Escherichia coli transformed with the cloned etf operon expressed MRHA and fimbriae that reacted with rabbit antiserum against the fimbrial major subunit of E. tarda, showing the implication of the fimbriae in the hemagglutination of E. tarda.

Key words: Edwardsiella tarda, mannose-resistant hemagglutination, fimbriae, gene clu

Identification of a Novel Chromosomal Region Associated with Infectious Hematopoietic Necrosis (IHN) Resistance in Rainbow Trout Oncorhynchus mykiss

<>Sok Kean Khoo1, 2, Akiyuki Ozaki2, 3, Fusayuki Nakamura4, Tetsuya Arakawa4,Shinichi Ishimoto4, Radoslav Nickolov5, Takashi Sakamoto2, Tetsuya Akutsu6,Mamiko Mochizuki6, Ikuo Denda7 and Nobuaki Okamoto2*<>1Laboratory of Cancer Genetics, Van Andel Institute, Grand Rapids, MI 49503, USA
2Department of Marine Bioscience, Tokyo University of Marine Science and
Technology, Tokyo 108-8477, Japan
3Department of Zoology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
4Mikawaichinomiya Station, Freshwater Resources Research Center,
Aichi Fisheries Research Institute, Hoi, Aichi 441-1222, Japan
5Department of Mathematical Sciences, Michigan Technological University,
Houghton, MI 49931, USA
6Fuji Trout Hatchery, Shizuoka Prefectural Fisheries Experimental Station,
Fujinomiya, Shizuoka 418-0108, Japan
7Nagano Prefectural Fisheries Experimental Station, Akashina,
Nagano 399-7102, Japan
(Received December 17, 2003)
ABSTRACT - Infectious hematopoietic necrosis (IHN) is an acute, systemic, rhabdoviral disease of salmonid species. To date, the effective approach in preventing or treating this epizootic disease is unknown. In this study, a genetic cross was established to elucidate the genetic markers associated with IHN resistance in rainbow trout Oncorhynchus mykiss. An IHN-resistant paternal strain of was crossed to a susceptible maternal strain to produce a F1 generation. Mating F1 males back to the maternal strain created F2, a backcross generation. Five backcross families were generated and IHNV was challenged on 92 individuals of each family. Linkage analysis was performed on the backcross families using 96 microsatellite markers from 25 linkage groups. LOD score as well as p values using the Pearson goodness of 2-test were calculated to identify the chromosomal regions associated with IHN resistance. The highest LOD score of 0.952 (= 0.39) was obtained in one backcross family. A novel chromosomal region (p = 0.011) on linkage group 2 consists of a cluster of 4 microsatellite markers, OmyFGT3TUF, OmyFGT25TUF, OmyRGT35TUF and BFRO0001, and they were identified as a potential region for quantitative trait loci of IHN resistance in rainbow trout.

Key words: infectious hematopoietic necrosis, IHN resistance, genetic linkage map, microsatellite marker, chromosomal region, Oncorhynchus mykiss, rainbow trout

Virulence of Viral Hemorrhagic Septicemia Virus (VHSV) Isolates from Japanese Flounder Paralichthys olivaceus in Rainbow Trout and Several Species of Marine Fish

<>Takafumi Ito1*, Koh-ichiro Mori2, Misao Arimoto2 and Kazuhiro Nakajima3<>1National Research Institute of Aquaculture, Fisheries
Research Agency, Tamaki, Mie 519-0423, Japan
2Kamiura Station, National Center for Stock Enhancement, Fisheries Research Agency, Kamiura,
Oita 879-2602, Japan
3Headquarters, Fisheries Research Agency,
Yokohama, 236-8648, Japan

(Received July 22, 2003)

ABSTRACT - Two Japanese isolates of viral hemorrhagic septicemia virus (VHSV), JF00Ehi1 (Genogroup I) and KRRV-9601 (Genogroup III), from Japanese flounder Paralichthys olivaceus were examined for the virulence to rainbow trout Oncorhynchus mykiss and 4 species of marine fish. Rainbow trout injected intraperitoneally with each isolate at a dose of 107.5 TCID50/fish showed neither death nor hemorrhage in the skeletal muscle, while the mortalities in Japanese flounder were both 100%. The mortalities by intramuscular injection with JF00Ehi1 at a dose of 107.0 TCID50/fish were 95.0%, 75.0% and 97.5% in black rockfish Sebastes inermis, red sea bream Pagrus major and yellowtail Seriola quinqueradiata, respectively.

Key words: VHSV, virulence, Paralichthys olivaceus, Oncorhynchus mykiss, experimental infection

Response of the Monogenean Neobenedenia girellae to Low Salinities

<>Naoko Umeda* and Noritaka Hirazawa<>Marine Biological Technology Center, Nippon Suisan Kaisha, LTD., Tsurumi-cho, Minamiamabegun,
Oita 876-1204, Japan

(Received October 3, 2003)

ABSTRACT - Effects of different salinities (8, 17, 24, 27, 30 and 34 ppt) on the egg laying, egg hatching and oncomiracidial swimming behavior of the monogenean Neobenedenia girellae were examined in vitro. Egg laying ability was affected at 8, 17, 24 ppt salinity and the number of laid eggs at these salinities were significantly lower than at 34 ppt salinity (control). Egg hatching rates at 8 and 17 ppt salinity were significantly lower than that of the control. The rate of non-motile oncomiracidia at 8 and 17 ppt salinity was significantly higher than that of the control. The results suggest that culturing fish in seawater with low salinity could be effective to control N. girellae
infections in aquaculture.

Key words: Neobenedenia girellae, salinity, oncomiracidium, Monogenia

Detection of Koi Herpesvirus in Koi Cyprinus carpio in Taiwan

<>Chien Tu1*, Ming-Chia Weng1, Jong-Rong Shiau2 and Shih-Yuh Lin1<>1Department of Biological Research, Animal Health Research Institute, Council of Agriculture, Tanshui 251, Taiwan, Republic of China
2Department of Epizootic Research, Animal Health Research Institute, Council of Agriculture, Tanshui 251, Taiwan, Republic of China

(Received October 17, 2003)

ABSTRACT - Mass mortality occurred in 2-year-old koi (fancy carp) Cyprinus carpio in Taiwan in 2002. Affected fish did not show any external signs except swollen gills. The consistent histological changes were observed in the gills; hyperplasia of epithelial cells, infiltration of eosinophilic granular cells and fusion of the secondary lamellae. Negatively stained nucleocapsids were icosahedral and 112 ± 1 nm in diameter. Koi herpesvirus (KHV) was detected in the diseased fish by PCR using specific primers for KHV, and sequence of the amplicon showed a 99% identity with the published data. This is the first report of KHV infection in Taiwan.

Key words: koi herpesvirus, KHV, histopathology, Cyprinus carpio, koi

Quality Control Ranges of Minimum Inhibitory Concentrations for Lactococcus garvieae and Photobacterium damselae subsp. piscicida

<>Michiko Kawanishi1*, Akemi Kojima1, Kanako Ishihara1, Hidetake Esaki1,Toshio Takahashi1, Shoko Suzuki1, Ikuo Hirono2, Takaji Iida3,Takashi Aoki2 and Yutaka Tamura1<>1National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries,
Kokubunji, Tokyo 185-8511, Japan
2Laboratory of Genetics and Biochemistry, Course of Aquatic Biosciences
Graduate School of Fisheries Science, Tokyo University of Marine
Science and Technology, Minato, Tokyo 108-8477, Japan
3National Research Institute of Aquaculture, Fisheries Research Agency,
Nansei, Mie 516-0193, Japan
(Received January 19, 2004)
ABSTRACT - It is necessary to determine the quality control ranges of the minimum inhibitory concentrations (MICs) for fish pathogenic bacterial strains in order to achieve comparability of test results. We derived tentative quality control ranges for Lactococcus garvieae and Photobacterium damselae subsp. piscicida (2 strains each), by using the standard agar dilution method of the National Committee for Clinical Laboratory Standards. We measured the MICs of 14 drugs for the 4 strains and 5 quality control strains under culture conditions recommended for both aquatic bacteria and determined tentative quality control ranges from the modal MIC ± 1 log2 dilution step.

Key words: Lactococcus garvieae, Photobacterium damselae subsp. piscicida, MIC, NCCLS.

Symposium Fish Disease Control under the Revised Pharmaceutical Administration and Regulations Law

<>Makoto Endo<>Tokyo University of Marine Science and Technology,
Tokyo 108-8477, Japan
ABSTRACT - The Pharmaceutical Administration and Regulations Law has been revised in Japan during 2003, resulting in a big confusion on fish disease control practice, because limited fish drugs and vaccines have been allowed to use in only a few fishes. This context motivated the Japanese Society of Fish Pathology to organize a symposium (4th/April/2004) at Kagoshima. The symposium was focused on the up-to-date disease control methods with respect to food safety, drug administration with scientific evidences, extension of available fish species in drug and vaccine administrations, responses of fish farming associations and pharmaceutical companies to the revised law. Finally, it was reconfirmed that the drug and vaccine administrations under the tight law and HACCP operations give the safety of farmed fish products to consumers.

Key words: disease control, food safety, drug and vaccine administrations, compliance